Role of salvage and phosphorylation in the immunostimulatory activity of C8-substituted guanine ribonucleosides

Lymphokine-like activity and selective stimulation of B cell growth is exerted by a group of synthetic ribonucleosides derivatized at C8 and exemplified by 8-bromoguanosine (8BrGuo), 8-mercaptoguanosine, and 7-methyl 8-oxoguanosine. However, relatively little is known about their molecular mechanism of action. Like naturally occurring nucleosides, 8BrGuo is taken up into lymphocytes by a process of facilitated diffusion. Naturally occurring nucleosides are then reclaimed by a well characterized salvage pathway, involving sequential phosphorolysis and phosphoribosylation. The studies reported in this communication demonstrate that, in contrast to naturally occurring nucleosides, 8BrGuo is not a substrate for salvage by purine nucleoside phosphorylase. The base that would be produced by putative phosphorolysis, 8-bromoguanine, is biologically inactive and is not a substrate for hypoxanthine-guanine phosphoribosyl-transferase. Accordingly, inhibitors of purine nucleoside phosphorylase-mediated salvage fail to inhibit nucleoside-induced immunostimulation selectively. Examination of the metabolism of 8BrGuo provides no direct evidence that 8BrGuo is phosphorylated by B lymphocytes. Direct enzymatic phosphorylation does not seem to be essential to the mechanism of action of the nucleoside insofar as competitive inhibition of deoxycytidine kinase (an enzyme that directly phosphorylates purines as well as pyrimidines) or of deoxyguanosine kinase fails to inhibit 8BrGuo stimulation selectively. Moreover, studies with synthetic nucleosides in which 3' and/or 5' hydroxyl groups were irreversibly blocked, precluding their phosphorylation, demonstrated that immunobiologic activity can occur in the absence of 3' and/or 5' phosphorylation. Finally, experiments with radiolabeled nucleosides provide no evidence to support the hypothesis that they are incorporated into cellular nucleic acid. These data, together with previous studies, suggest that it is the unmetabolized nucleoside that is active and, as such, is most likely to act in a regulatory capacity.     

Ligand binding sites for a synthetic B cell growth and differentiation factor

The mechanism of action of a group of synthetic lymphokine-like molecules, the C8-substituted guanine ribonucleosides, was studied. Among their pleiotropic effects on B cells are the increased expression of surface Ia antigens, induction of polyclonal immunoglobulin secretion, enhancement of thymus-dependent as well as thymus-independent antibody responses, and transmission of T cell-like differentiative signals to B cells. However, relatively little is known about their molecular mechanism of action. In the current article, the interaction of 8-bromo-guanosine (8BrGuo), a prototypical C8-substituted guanine ribonucleoside, with cellular components was examined. Rapidly exchangeable (free) and slowly exchangeable (bound) 8BrGuo pools exist within B cells. The bound nucleoside pool loses its ability to be retained by a boronate affinity resin (despite its resistance to metabolic processing) and localizes to the cytosol on sucrose density gradients. Binding affinity, ligand specificity, and cellular specificity of binding all correlate closely with observed functional properties of these molecules. Together, these data suggest that the binding interaction mediates the biologic activities of 8BrGuo, and that the binding site acts as a functional nucleoside receptor.     

Active transformation of tolerogenic to immunogenic signals in T and B cells by 8-bromoguanosine

The injection of deaggregated human gamma-globulin (DHGG) into A/J mice results in the establishment of a state of unresponsiveness to subsequent challenge with immunogenic aggregated human gamma-globulin (AHGG). Administration of the B cell activator 8-bromoguanosine (8BrGuo) 3 hr after administration of DHGG converts the tolerogen to an immunogen and results in an antibody response of even greater magnitude than the primary response elicited by AHGG alone. Adoptive transfer studies with separated populations of T and B cells demonstrated that although transformation of the tolerogenic signal to an immunogenic signal involves effects of 8BrGuo on both T cells and B cells, the major effect appears to be activation of antigen-specific T cells that would otherwise become tolerant. Modulation of T cell tolerance could conceivably be mediated either by direct or indirect mechanisms. Interestingly, optimal responsiveness of B cells from animals treated with DHGG and 8BrGuo is not a T cell-independent event, but requires antigen-reactive T cells. 8BrGuo is not able to override unresponsiveness when given 10 to 20 days after tolerance induction, at a time point when both T and B lymphocytes are tolerant. However, when given at day 60, when T cells (but not B cells) remain tolerant to this antigen, the nucleoside is able to terminate the tolerant state prematurely, possibly by providing an alternate T helper-like signal directly to B cells or by recruiting nonspecific functional T helper cells.     

Purine nucleoside modulation of functions of human lymphocytes.

The accumulation of endogenous substrates in patients with adenosine deaminase deficiency or purine nucleoside phosphorylase deficiency is believed to be responsible for the immunodeficiency observed in these patients. To identify the lymphocyte populations that are most susceptible to these substrates, we investigated the effect of their nucleoside analogs on a number of T and B cell functions of human lymphocytes. We found that tubercidin (Tub), 2-chloro 2'deoxyadenosine (2CldA), 2-fluoro adenine arabinoside-5'phosphate (FaraAMP), and 9-beta-D-arabinosyl guanine (AraGua) inhibited the proliferative responses of human peripheral blood mononuclear cells (PBMC) to polyclonal activators (PHA, OKT3 mab) or to allogeneic PBMC in mixed lymphocyte cultures (MLC). Addition of recombinant IL-2 from the beginning of the culture did not alter the inhibition by Tub of the proliferative responses of PBMC. These purine nucleoside analogs also inhibited the proliferative responses of purified human peripheral blood CD4+ and CD8+ T cells to PHA and of purified B cells to SAC. The concentrations of these nucleosides required to achieve a given degree of inhibition of proliferative responses of T lymphocyte subpopulations or B cells was similar, suggesting that these analogs do not exhibit any selectivity for these purified lymphocyte populations. Tub and FaraAMP, respectively, inhibited and enhanced, at the effector phase, both NK cytotoxicity and specific T cell-mediated cytotoxicity. In contrast to these findings, LAK cytotoxicity at the effector phase was not significantly inhibited by Tub, and was not enhanced by FaraAMP. Both analogs inhibited rIL-2-induced proliferative responses of PBMC, but did not affect the generation of LAK cytotoxicity (induction phase) against the K562 targets when added at the beginning of the culture. This suggests that DNA synthesis is not required for LAK cell induction. Both Tub and FaraAMP inhibited immunoglobulin production (IgG and IgM) by PBMC in the PWM-induced system. These results demonstrate that purine nucleoside analogs significantly inhibited a number of functions of human lymphocytes. Although selectivity for T lymphocyte subpopulations and B cells was not observed, a differential effect of Tub and FaraAMP on LAK cytotoxicity versus NK cytotoxicity and specific T cell cytotoxicity was found.     

Carbon monoxide inhibits T lymphocyte proliferation via caspase-dependent pathway

T lymphocyte activation and proliferation is involved in many pathological processes. We have recently shown that carbon monoxide (CO), an enzymatic product of heme oxygenase-1 (HO-1), confers potent antiproliferative effects in airway and vascular smooth muscle cells. The purpose of this study was to determine whether CO can inhibit T lymphocyte proliferation and then to determine the mechanism by which CO can modulate T lymphocyte proliferation. In the presence of 250 parts per million CO, CD3-activated T lymphocyte proliferation was, remarkably, inhibited by 80% when compared with controls. We observed that the antiproliferative effect of CO in T lymphocytes was independent of the mitogen-activated protein kinase or cGMP signaling pathways, unlike what we demonstrated previously in smooth muscle cells. We demonstrate that CO inhibited caspase-3 and caspase-8 expression and activity, and caspase inhibition with benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-FMK pan-caspase inhibitor) blocked T lymphocyte proliferation. Furthermore, in caspase-8-deficient lymphocytes, the antiproliferative effect of CO was markedly attenuated, further supporting the involvement of caspase-8 in the antiproliferative effects of CO. CO also increased the protein level of p21(Cip1), and CO-mediated inhibition of caspase activity is partially regulated by p21(Cip1). Taken together, these data suggest that CO confers potent antiproliferative effects in CD3-activated T lymphocytes and that these antiproliferative effects in T lymphocytes are mediated by p21(Cip1)-dependent caspase activity, in particular caspase-8, independent of cGMP and mitogen-activated protein kinase signaling pathways.     

Autologous mixes lymphocyte reaction in human cord blood lymphocytes: decreased generation of helper and cytotoxic T-cell functions and increased proliferative response and induction of suppressor T cells

T lymphocytes from neonates proliferated significantly more than peripheral blood T lymphocytes from adults in autologous mixed lymphocyte reactions (AMLR). AMLR-activated cord, as compared to adult T lymphocytes, exerted significantly less nonspecific cytotoxic activity on PHA-stimulated adult mononuclear cells and Epstein-Barr virus-transformed target cells. The impaired generation of cytotoxicity of cord T cells was not corrected by Interleukin-2. Blood T lymphocytes from adults activated in AMLR synthesized a helper factor that supported PWM-induced proliferation and immunoglobulin production in both adult and cord B lymphocytes. In contrast, cord blood T lymphocytes failed to produce the helper factor for B lymphocytes. T cells from AMLR cultures established with neonatal lymphocytes showed suppressor activity, as assessed in PWM-stimulated immunoglobulin synthesis of adult peripheral-blood mononuclear cells, significantly higher than that exhibited by T cells from AMLR cultures performed with lymphocytes from adults. Finally, neonatal B lymphocytes could be activated to the production of IgM but not IgG by either adult AMLR-derived helper factor plus PWM or by Epstein-Barr virus, whereas adult B cells secreted both IgM and IgG under the same type of stimulation.     

The immune response modifiers imiquimod and R-848 are potent activators of B lymphocytes

Imiquimod and R-848 are members of a family of immune response modifiers that stimulate cytokine production in monocyte/macrophages and dendritic cell cultures. This study evaluated the effects of the imidazoquinolines, imiquimod and R-848, on B lymphocyte activation. Both agents induced proliferation of murine T-cell-depleted and highly purified splenic B cell preparations as well as purified human B cells. Resting and activated B cells responded to these agents, with activated cells responding more efficiently. B cells from the LPS-hyporesponsive C3H/HeJ mice and guanosine-hyporesponsive SJL mice proliferated in response to imiquimod and R-848, indicating a different mechanism of action than lipopolysaccharide and guanine nucleosides. B cells were also stimulated by imiquimod and R-848 to produce increased immunoglobulin levels. Increased expression of a number of B cell activation markers were seen following imiquimod or R-848 stimulation. Finally, R-848 was shown to act as a vaccine adjuvant enhancing OVA-specific IgG2a levels while suppressing total IgE. These results indicate that R-848 and imiquimod are potent activators of B lymphocytes and are capable of augmenting antigen-specific immunoglobulin production.     

Bromination of guanosine and cyclic GMP confers resistance to metabolic processing by B cells

The metabolic fates of 8-bromoguanosine (8BrGuo) and 8-bromoguanosine-3'5'-cyclic monophosphate (8Br-cGMP) were examined in cultures of murine B lymphocytes. These compounds exert striking immunostimulatory effects upon bone marrow-derived lymphoid cells in vitro. Both 8BrGuo and 8Br-cGMP were resistant to metabolic processing by these cells. That purine metabolic pathways are intact and operant in B cells was demonstrated by the ready degradation and phosphorylation of native guanosine and cyclic GMP. Inaccessibilty of the substrate to the relevant enzymes was ruled out as an explanation by the observation that the brominated compounds also were resistant to processing in broken cell preparations. Moreover, 8BrGuo did not interfere with the cellular machinery for metabolizing native guanosine. The implications of these observations for studying the actions of purine nucleotides, cyclic nucleotides, and their enzymatic processing in B cells are discussed.     

Human epidermal cells from ultraviolet light-exposed skin preferentially activate autoreactive CD4+2H4+ suppressor-inducer lymphocytes and CD8+ suppressor/cytotoxic lymphocytes

In vivo exposure of human epidermis to UV abrogates the function of T6+DR+ Langerhans cells and induces the appearance of Ag-presenting T6-DR+ OKM5+ cells in the epidermis. Since UV exposure of murine skin results in Ts lymphocyte activation, we investigated the capacity of human epidermal cells (EC) harvested 3 days after in vivo UV exposure to activate regulatory and effector autologous T lymphocyte subsets. T lymphocytes were separated into CD8+ suppressor/cytotoxic lymphocytes and CD4+ helper/inducer lymphocytes by C lysis and panning. The CD4+ subset was further divided by using the 2H4 mAB to obtain CD4+2H4+ lymphocytes (inducers of TS lymphocytes) and CD4+2H4- lymphocytes (inducers of B cell Ig production and inducers of cytotoxic T cells). Unirradiated suction blister-derived EC from control skin (C-EC) and from skin exposed in vivo to UV (UV-EC) were cultured with purified autologous T lymphocyte subsets in the absence of added Ag. The resultant T lymphocyte proliferation was detected by [3H]thymidine uptake. UV-EC were highly effective in the stimulation of CD4+ lymphocytes, whereas C-EC were poor stimulators. The stimulator effect of UV-EC was abrogated after depletion of DR+ UV-EC. When CD4+ lymphocytes were fractionated, UV-EC consistently demonstrated enhanced ability to stimulate suppressor-inducer CD4+2H4+ lymphocytes relative to C-EC. Although less responsive than CD4+2H4+ lymphocytes, CD4+2H4- lymphocytes also demonstrated greater proliferation to UV-EC than to C-EC. Neither UV-EC nor C-EC were able to activate CD8+ lymphocytes devoid of CD4+ lymphocytes. However, after addition of rIL-2 at concentrations that allow binding only to the high affinity IL-2R on T lymphocytes, UV-EC induced vigorous proliferation of CD8+ lymphocytes, whereas C-EC induced only background levels of proliferation. C lysis of leukocytes resident within UV-EC resulted in 66 to 70% reduction of CD8+ lymphocyte proliferation. In conclusion, UV-EC may activate CD8+ lymphocytes by at least two pathways: (1) UV-EC activation of CD4+2H4+ lymphocytes may induce differentiation/proliferation of CD8+ suppressor cells and (2) UV-EC activation of CD4+ cells may induce IL-2 production, that, in combination with UV-induced epidermal leukocytes, stimulates CD8+ cells.     

Folate deficiency inhibits the proliferation of primary human CD8+ T lymphocytes in vitro

Folate is required for one-carbon transfer reactions and the formation of purines and pyrimidines for DNA and RNA synthesis. Deficiency of folate can lead to many clinical abnormalities, including macrocytic anemia, cardiovascular diseases, birth defects, and carcinogenesis. The nucleotide imbalance due to folate deficiency causes cell cycle arrest in the S phase and uracil misincorporation into DNA, which may result in DNA double-strand breaks during repair. The role of folate in the immune system has not been fully characterized. We cultured PHA-activated human T lymphocytes in varying concentrations of folate, and measured proliferation, cell cycle, apoptosis, uracil misincorporation, and proportions of Th cells (CD4(+)) and cytotoxic T (CD8(+)) cells. Folate deficiency reduced proliferation of T lymphocytes, induced cell cycle arrest in the S phase, induced apoptosis, and increased the level of uracil in DNA. Folate deficiency also increased the CD4(+) to CD8(+) ratio due to a marked reduction of CD8(+) cell proliferation. Folate or nucleoside repletion of folate-deficient cells rapidly restored T lymphocyte proliferation and normal cell cycle, reduced the DNA uracil content, and lowered the CD4(+) to CD8(+) ratio. These data suggest that folate status may affect the immune system by reducing the capacity of CD8(+) cells to proliferate in response to activation.     

Activation of lymphocytes by a thiol-derivatized nucleoside: characterization of cellular parameters and responsive subpopulations

Lymphocyte activation, whether specific or nonspecific, is generally conceptualized as initiated by the binding of an activating ligand to a surface membrane receptor, followed by transduction of the signal across the cell membrane. In many situations several qualitatively distinct signals are required. We have recently described a new class of lymphocyte activator, the C8 bromine substituted guanine ribonucleosides, that traverse the cell membrane, bypassing classical triggering mechanism(s), apparently activating the lymphocyte at an intracellular site. However, the identity of the lymphocyte population(s) activated, as well as the nature of any cellular interactions involved in activation, has not been studied heretofore. The present experiments describe the cellular parameters of lymphocyte activation by a thiol substituted member of this class of activators, 8-mercaptoguanosine (8MGuo). Upon addition of this nucleoside derivative to cultures of murine spleen cells, a marked increase in [3H]TdR uptake and blast transformation ensues. Normal splenic B cells and spleen cells from congenitally athymic (nu/nu) mice are responsive to 8MGuo, whereas thymocytes and splenic T cells are not. Two subpopulations of B cells appear to be involved in the response to this nucleoside. The predominant one is a mature population that bears surface delta-chains, la antigens, C receptors, and (by indirect evidence) the Lyb3, 5, and 7 antigens. These cells also bear mu-chain and Fc receptors. In addition, a second, minor subpopulation of less mature cells that bear only mu-chain and Fc receptors also appears to be reactive to 8MGuo. The existence of this immature, reactive B cell subset was confirmed by observation of 8MGuo responsiveness in lymphocytes from 4-day-old mice whose cells do not yet exhibit these later-appearing markers. Accessory cells appear to play a minimal, if any, role in the 8MGuo response. These results establish two distinct B cell subpopulations as the major and minor cellular targets of C8-derivatized nucleosides, and suggest that the activation process results from a direct interaction between the nucleoside and target cell.     

连翘酯苷对小鼠脾脏淋巴细胞体外增殖与分泌功能的影响

目的分析连翘酯苷（FS）对小鼠脾脏T和B淋巴细胞增殖、分泌NO和TNF-α的影响,初步探讨其免疫调节作用机制。方法无菌操作分离小鼠脾脏,制备脾脏细胞并用含10%胎牛血清的RPMI 1640培养,在培养液中分别加入刺激剂刀豆蛋白（ConA）和脂多糖（LPS）以及不同浓度40、80、160μg/mL的FS共培养不同时间,采用MTT法检测T和B淋巴细胞的吸光度变化,ELISA和Griess法分别检测细胞分泌TNF-α和NO的水平。结果低浓度和中浓度FS对ConA诱导T淋巴细胞24 h和48 h后细胞增殖和存活率明显提高,诱导时间延长至72 h后FS明显抑制细胞转化;低浓度FS对LPS诱导脾脏B淋巴细胞24 h后细胞增殖和生存率显著提高;FS促进小鼠脾脏T和B淋巴细胞分泌NO;FS促进B淋巴细胞分泌TNF-α,中浓度FS促进T淋巴细胞分泌TNF-α而高浓度反而抑制其分泌。此外,FS对环磷酰胺（CY）处理小鼠的脾脏淋巴细胞体外增殖有明显影响,对细胞NO分泌影响不显著。结论结果提示FS可能通过影响小淋巴细胞增殖和细胞因子分泌而调节免疫细胞功能。     

Effects of ascorbic acid and sodium ascorbate on cyclic nucleotide metabolism in human lymphocytes.

L-ascorbic acid (LAA) augmented cGMP many-fold in highly purified human peripheral blood lymphocytes. The cGMP response occurred within 10 sec and persisted for at least 60 min. D-ascorbic acid (DAA) and dehydroascorbic acid (DHAA) were also equally active in enhancing cGMP concentrations but metabolic precursors of ascorbic acid and other inorganic acids did not increase cGMP levels. Determination of the amount of DHAA contaminating the LAA precluded the possibility that it was solely responsible for the enhanced cGMP levels. The sodium or calcium salts of ascorbic acid did not increase cGMP concentrations. If these neutralized preparations were acidified, increased cGMP concentrations were then noted. In broken cell preparations, LAA, DAA, and DHAA and to a lesser extent sodium ascorbate (NaA) enhanced guanylate cyclase activity while neither inhibited cAMP or cGMP phosphodiesterase (PDE) activity. The possible role of H2O2, fatty acid liberation, prostaglandin production, oxidizing-reducing agents, and free radical formation in mediating the effects of ascorbic acid on cGMP levels were evaluated, but none of these potential mechanisms were definitively proven to be a required intermediary for the cGMP enhancing activity of ascorbic acid. LAA, DHAA or NaA did not induce lymphocyte transformation or modulate lectin-induced mitogenesis.     

Development and regulation of chlamydia-responsive murine B lymphocytes

We have examined characteristics of chlamydia-stimulated mouse B cells as well as cells that regulate polyclonal responses in vitro. B lymphocyte proliferation stimulated by chlamydia arises at a similar time as Escherichia coli lipopolysaccharide (LPS)-induced proliferative responses during ontogeny. In contrast, development of immunoglobulin (Ig)-secreting cells after chlamydia stimulation is delayed by several weeks relative to ontogeny of LPS-inducible plaque-forming cells (PFC). The lack of Ig secretion by immature B cells is not due to a deficiency of Lyb5+ B lymphocytes, since X-linked immunodeficient (xid) NBF1 mice that lack this B lymphocyte population respond well to chlamydia stimulation. Adherent cells are important for chlamydia-stimulated B lymphocyte differentiation, but are not as necessary for their proliferation. Neither adult adherent cells nor T cells can correct the inability of immature spleen cells to develop into Ig-secreting cells; spleen cells from 2-wk-old mice (i.e., immature B cells) will not suppress adult B lymphocyte responses to chlamydia. When B lymphocytes are separated according to their buoyant densities, chlamydia stimulates low density (activated) B cells to proliferate and differentiate better than high density (resting) cells. Proliferative responses to chlamydia arise earlier during ontogeny, do not require adherent cells, and can proceed to a relatively greater extent in resting B cell population (compared with activated B cells) than induction of Ig-secreting cells.     

B lymphocyte production in the bone marrow of mice with X-linked immunodeficiency (xid)

CBA/N mice carry an X-linked recessive immunodeficiency (xid) gene manifested by the absence of a B lymphocyte subpopulation, but the manner in which the xid gene exerts its effect on B lymphocyte development is unknown. The production of B lymphocytes in the bone marrow of CBA/N mice has now been compared with that of normal CBA/J mice by using two in vivo assays: immunofluorescence stathmokinetic studies measured pre-B cell proliferation, whereas radioautographic [3H]thymidine labeling was used to evaluate small lymphocyte turnover. Although the total cellularity of CBA/N mouse bone marrow was greater than normal, the absolute number of marrow small lymphocytes, pre-B cells, and B lymphocytes were all similar to those in CBA/J controls. Furthermore, in the bone marrow of CBA/N mice, the proliferation rate of pre-B cells, calculated from their rate of entry into mitosis, and the turnover rate of small lymphocytes, derived from their rate of [3H]thymidine labeling, were not significantly different from those seen in nondefective mice. The present findings that pre-B cell proliferation and small lymphocyte production proceed at similar rates in the bone marrow of xid and normal mice suggest that the xid gene does not act at the level of primary B cell genesis in the bone marrow. The findings are in accord with the view that the xid gene produces a maturation block or a functional abnormality among B lymphocytes in the peripheral lymphoid tissues rather than the deletion of a sublineage of B lymphocytes in the bone marrow.     

Immunomodulatory role of 1,25-dihydroxyvitamin D3.

The active vitamin D metabolite 1,25-dihydroxyvitamin D3 [1,25-D3] is thought to promote many of its actions through interaction with a specific intracellular receptor. The discovery of such receptors in monocytes and activated lymphocytes has led investigators to evaluate the role of the hormone on the immune system. The sterol inhibits lymphocyte proliferation and immunoglobulin production in a dose-dependent fashion. At a molecular level, 1,25-D3 inhibits the accumulation of mRNA for IL-2, IFN-gamma, and GM-CSF. At a cellular level, the hormone interferes with T helper cell (Th) function, reducing Th-induction of immunoglobulin production by B cells and inhibiting the passive transfer of cellular immunity by Th-clones in vivo. The sterol promotes suppressor cell activity and inhibits the generation of cytotoxic and NK cells. Class II antigen expression on lymphocytes and monocytes is also affected by the hormone. When given in vivo, 1,25-D3 has been particularly effective in the prevention of autoimmune diseases such as experimental autoimmune encephalomyelitis and murine lupus but its efficacy has been limited by its hypercalcemic effect. Synthetic vitamin D3 analogues showing excellent 1,25-D3-receptor binding but less pronounced hypercalcemic effects in vivo have recently enhanced the immunosuppressive properties of the hormone in autoimmunity and transplantation.     

Mice lacking the CARD of CARMA1 exhibit defective B lymphocyte development and impaired proliferation of their B and T lymphocytes

CARMA1 (originally called CARD11) is a membrane-associated guanylate kinase family member that is required for T cell receptor (TCR)-induced NF-kappa B activation in T cell leukemia lines. It uses its N-terminal caspase activation and recruitment domain (CARD) to interact with the CARD in the downstream adaptor Bcl-10. We show that primary B and T lymphocytes from knock-in mice expressing only a CARDless form of CARMA1 (Delta CARD) are defective at mitogen-induced NF-kappa B activation and fail to proliferate. CARMA1 mutant mice exhibited normal T but impaired B cell development; CD5(+) peritoneal B cells were absent, and serum immunoglobulin levels were markedly reduced. A lacZ reporter gene knocked into the CARMA1 locus confirmed lymphocyte-specific expression of CARMA1. Thus, CARMA1 has an essential role in mediating B and T lymphocyte proliferation and requires its CARD to engage downstream signaling components.     

B lymphocytes from hyporesponsive SJL mice contain aberrant nucleoside binding sites

C8-substituted guanine ribonucleosides activate B cells by a novel pathway that apparently is independent of GTP-binding proteins and protein kinase C. B lymphocytes from SJL mice are hyporesponsive to antigen-independent inductive signals transmitted by these nucleosides. In the current studies, the basis for this observation was explored. Responses of normal murine strains to these agents have been dissociated into antigen-independent (inductive) and antigen-dependent (differentiative) types by use of the 7,8-disubstituted guanine ribonucleosides. Dose-response profiles for inductive responses appear to correlate with apparent Kd values for low-affinity nucleoside binding sites; dose-response curves for antigen-dependent differentiative responses correlate with apparent Kd values for high-affinity binding sites. It was found that the SJL low-affinity site exhibits an apparent Kd that is approximately 10- to 20-fold lower in affinity for 8BrGuo than that of normal CBA mice. Although the low-affinity site in normal murine strains displays nearly equivalent affinity toward C8-substituted and 7,8-disubstituted nucleosides, the low-affinity site of SJL mice binds 7,8-disubstituted compounds with approximately 5-fold higher affinity than it does monosubstituted compounds. The dissociation constant for high-affinity nucleoside binding sites of SJL mice was only slightly different from that of CBA mice, consistent with the observation of essentially normal antigen-dependent nucleoside-mediated activity in SJL mice. The current observations support (a) a role for low-affinity binding sites in antigen-independent inductive events, (b) a role for high-affinity binding sites in antigen-dependent differentiative events mediated by substituted guanine nucleosides, and (c) the existence of aberrant low-affinity binding sites in B cells from SJL mice.     

Cyclic nucleotides and calcium ions in the activation of B-lymphocyte movement in mice by an anti-immunoglobulin serum

It was shown that B lymphocyte motility activated by anti-immunoglobulin serum may be abrogated in a Na+-deficient medium and in a 10(-5)M trifluoperazine-containing medium but not in a Ca2+-deficient medium. The tetracycline fluorescence test demonstrated Ca2+ efflux from isolated B lymphocyte mitochondria due to Na+ exposure. The radioimmunoassay demonstrated the cGMP level to rise after exposure to anti-immunoglobulin serum. The Na+-dependent Ca2+ efflux from the mitochondria might be the main mechanism in anti-immunoglobulin serum activation of B lymphocytes and in the cGMP level rising.     

Levamisole and bovine immunity: in vitro and in vivo effects on immune responses to herpesvirus immunization

Levamisole was shown to enhance in vitro blastogenic responses of bovine lymphocytes to nonspecific mitogens (phytohemagglutinin and pokeweed mitogen) as well as to infectious bovine rhinotracheitis virus and purified protein derivative. Greatest enhancement was observed at suboptimal concentrations of viral antigen. In addition to enhancing lymphocyte reactivity levamisole also affected macrophage activity as determined by increased Fc receptor activity and [3H]glucosamine incorporation. Levamisole (5-50 micrograms/mL) enhanced type II immune (or gamma) interferon production by macrophage-lymphocyte cultures. Administration of levamisole and attenuated infectious bovine rhinotracheitis vaccine virus in vivo did not elevate cellular or humoral responses.